1. Mass spectrometry-based proteomics Jeff Johnson Feb 19, 2014

2. MS Proteomics in a Nutshell Ionization – Delivering macromolecules to the MS Ion Manipulation – Trapping and ejecting analytes of interest Fragmentation – Breaking apart for more information Mass analysis and detection – Measuring masses and quantifying intensities

3. MS Proteomics in a Nutshell Ionization – Delivering macromolecules to the MS Ion Manipulation – Trapping and ejecting analytes of interest Fragmentation – Breaking apart for more information Mass analysis and detection – Measuring masses and quantifying intensities

4. Macromolecular Ionization for MS Analyte must be in the gas phase for mass analysis Analyte must be charged in order to be manipulated by electric and magnetic fields – All mass analyzers measure mass-to-charge ratios (m/z) Two predominant approaches (shared the Nobel prize in 2002) – Matrix assisted laser desorption ionization – Electrospray ionization

5. MALDI Ionization Sample is spotted in a matrix that readily absorbs UV/IR light and is vaporized by a laser – Common matrix: 2,5- dihydroxybenzoic acid (DHB) Advantages – Fast and easy – Spots can be reanalyzed later – Most analytes get one +ive charge makes it easy to deconvolute Disadvantages – Harsh. Often breaks analytes apart (e.g., breaks phosphorylation) – Not easily combined with online HPLC separations

6. Electrospray Ionization Sample is dissolved in liquid and pushed through a charged needle and sprayed into an evaporation chamber – Droplets pulled into the MS source by electric potential between the needle and the MS – Heated ion transfer tube evaporates water molecules in droplets leaving +ively charged analytes in the gas phase Advantages – Compatible with online HPLC separations – “Soft” ionization maintains label and non- covalent interactions Disadvantages – Analytes can have different numbers of charges, can be difficult to deconvolute without high mass accuracy – Different samples going through the same electrospray tip causes carryover problems Especially bad with online HPLCs

7. Ionization is Nearly Impossible to Predict 2x A B A B X Different molecules ionize with different efficiencies and are very difficult to predict MS intensity ratios between different molecules do not reflect ratios in the sample from which they were derived Most quantification by MS is relative

8. Ionization is Nearly Impossible to Predict Different molecules ionize with different efficiencies and are very difficult to predict MS intensity ratios between different molecules do not reflect ratios in the sample from which they were derived Most quantification by MS is relative A A A A Sample 1Sample 2 Sample 1Sample 2 * Assumption: MS run 1 = MS run 2

9. MS Proteomics in a Nutshell Ionization – Delivering macromolecules to the MS Ion Manipulation – Trapping and ejecting analytes of interest Fragmentation – Breaking apart for more information Mass analysis and detection – Measuring masses and quantifying intensities

10. Ion Manipulation We need a way to select only ions of interest – Most detectors are just electron multipliers that don’t measure mass but just detect a thing hitting the multiplier – We can manipulate ions to deliver defined mass ranges to the detector to get a mass spectrum Two common tools: – Ion traps – Quadrupoles – Both use electric and magnetic fields to select ions of a particular m/z range

11. Ion Trap Ions are trapped by 3D electric field by DC and AC applied to the electrodes An ion trap can accumulate ions as they come in from the source and store them Low resolution: +/- 1 Da

12. Quadrupole Can be thought as a mass filter DC and AC fields applied that stabilize a trajectory for ions in a desired mass range, undesired ions are ejected Quadrupole operate with a continuous flow of ions Low resolution (+/- 1 Da)

13. MS Proteomics in a Nutshell Ionization – Delivering macromolecules to the MS Ion Manipulation – Trapping and ejecting analytes of interest Fragmentation – Breaking apart for more information Mass analysis and detection – Measuring masses and quantifying intensities

14. Fragmentation Usually measuring the mass of an analyte is not enough to conclusively identify it By fragmenting an analyte and measuring the masses of the fragments we can obtain further information to identify the analyte There are many types of fragmentation but collision-induced dissociation (CID) is the most common – Fastest and most generally successful for the widest variety of proteins and peptides

15. Collision-Induced Dissociation Give ions kinetic energy and collide with gas molecules (He) Collisions build up potential energy until a fragmentation event can occur Ideally potential energy is strong enough to break a single peptide bond but not strong enough to fragment further Can be done in an ion trap or a quadrupole

16. Collision Induced Dissociation AEPTIR H2OH2O Fragment (somewhat) randomly along the peptide backbone

17. M/z Intensity AEP A AE AEPT 72.0 201.1 298.1 399.2 B-type Ions

18. M/z Intensity RITPEA H2OH2O Y-type Ions

19. M/z Intensity RITPEA H2OH2O B-type, A-type, Y-type Ions

20. MS Proteomics in a Nutshell Ionization – Delivering macromolecules to the MS Ion Manipulation – Trapping and ejecting analytes of interest Fragmentation – Breaking apart for more information Mass analysis and detection – Measuring masses and quantifying intensities

21. Mass Analysis and Detection Magnetic Sector MS All mass analyzers achieve the same thing: physical separation based on mass:charge Magnetic sector is the simplest and one of the earliest types

22. FT-ICR MS FT-ICR = Fourier transform – ion cyclotron resonance Ion injected in line with a strong magnetic field that induces a cyclical motion Radius of the cyclotron motion is proportional to m/z

23. Time-of-flight MS Medium / High Resolution

24. Quadrupole and Ion Trap MS Electron multiplier You can use a quadrupoles or ion traps to “scan out” ions across an entire mass range to a detector by gradually ramping voltages Low resolution but electron multipliers make these very sensitive

25. Orbitrap MS Characteristic frequencies: – Frequency of rotation ω φ – Frequency of radial oscillations ω r – Frequency of axial oscillations ω z r z φ

26. Power of Fourier Transforms FTs convert from time domain to freq domain Instead of a single measurement the m/z is measured over a period of time and the FT essentially averages all those measurements Resulting data is very high resolution

28. Chromatography to Simplify Complexity Complexity hurts sensitivity A constant, defined number of ions can be analyzed in each MS scan Sensitivity is constant (around 1 fmol) A scan with fewer ions is more sensitive than a scan with many Complex Sample MS

29. Chromatography to Simplify Complexity C18 RP column ACN gradient A B C D A B C D Complex Sample HPLC MS

30. Chromatography to Simplify Complexity Very Complex Sample Online HPLC (RP) MS Offline HPLC (e.g., SCX) SCX Fractions Injected individually

31. Acquiring MS Data Data acquisition depends on experimental goals – Data-dependent acquisition MS attempts to acquire data to allow you to identify a maximum number of unknowns Commonly used for analyses where you don’t know what you’re looking for – Targeted acquisition MS only acquires data for what you tell it to acquire Much more sensitive than data-dependent, but also more limited in scope

32. Data-Dependent Acquisition

33. High resolution survey scan (<5 ppm mass accuracy) 1 2 3

34. Data-Dependent Acquisition Low resolution MS/MS scan 1

35. Data-Dependent Acquisition Low resolution MS/MS scan 2

36. Data-Dependent Acquisition Low resolution MS/MS scan 3

37. Peptide Identification AA sequence DB (Species UniProt)

38. Peptide Identification 1 1 2 2 3 3 AA DBs restricted by parent ion mass measured in survey scan AA sequence DB (Species UniProt)

39. Peptide Identification MS/MS 1 MS/MS 2 MS/MS 3 1 1 2 2 3 3 AA DBs restricted by parent ion mass measured in survey scan AA sequence DB (Species UniProt)

40. Probabilistic Matching (X!Tandem) by-Score= Sum of intensities of peaks matching B-type or Y-type ions HyperScore= Hyper Score # of Matches Best Hit Second Best

41. Model spectrum comparisons

42. Pattern Matching (Sequest)

43. Sequest XCorr Cross Correlation (direct comparison) Auto Correlation (background) XCorr = Offset (AMU) Correlation Score

44. Targeted Acquisition with a QQQ A priori knowledge required: SRM assay development for a list of proteins/peptides of interest  Information derived from label-free unbiased proteomic analysis SRM Assay

45. “Sensitivity” Sensitivity of a MS is well defined, but the ability to identify something is a very different concept – Ability to detect depends on: Sample complexity MS sensitivity MS speed – A faster MS can collect go deeper in each survey scan – Think “top 10” vs. “top 50” MS mass accuracy – Better mass accuracy improves the ability to identify peptides but sacrifices speed and MS sensitivity – Especially important for variable modifications – The “best” method is very dependent on the experimental goals

46. Database Searching Ion trap +/- 1 Da Orbitrap +/- 0.002 Da Database “search space”

47. Database Searching Ion trap +/- 1 Da Orbitrap +/- 0.002 Da Database “search space” +S/T/Y phosphorylation

48. Database Searching Ion trap +/- 1 Da Orbitrap +/- 0.002 Da Database “search space” +S/T/Y phosphorylation

49. Protein Quantification A mass spectrometer is an inherently quantitative device but the ionization source is not – Different peptides/proteins are ionized with drastically different efficiencies – Absolute abundances in a mass spectrometer are not precisely indicative of abundance in a sample Solution: stable isotope labeling – Compare samples that have been labeled with stable isotopes ( 13 C, 15 N, 2 H) – ‘Heavy’ isotopes behave chemically identically to their ‘light’ counterparts but are separated in the MS

50. Isotope Coded Affinity Tag (ICAT)

53. Stable Isotope Labeling of Amino Acids in Culture (SILAC) Grow cells in media supplemented with stable isotope-labeled amino acids Combine samples at the level of cells and process as one sample Minimize variability between samples for lysis and digestion Different samples separated by mass in the MS

54. Absolute Quantification (AQUA)